Lac expression system

Abstract

Provided herein is a nucleic acid comprising a mutant lac operator operably linked to a gene of interest, a host cell comprising the nucleic acid, and a method of using the host cell to express the gene of interest. Also provided is a recA-mediated cloning method.

1 . A nucleic acid comprising a mutant lac operator operably linked to a gene of interest, wherein the mutant lac operator has increased affinity for a LacI repressor protein. 
     
     
         2 . The nucleic acid of  claim 1 , wherein the nucleic acid is a plasmid. 
     
     
         3 . The nucleic acid of  claim 1 , wherein the nucleic acid is a chromosome. 
     
     
         4 . The nucleic acid of  claim 1 , wherein the increased affinity is at least 5-fold. 
     
     
         5 . The nucleic acid of  claim 4 , wherein the sequence of the mutant lac operator comprises any one of SEQ ID NOS: 2-6. 
     
     
         6 . The nucleic acid of  claim 1 , wherein the gene of interest is T7 gene 1. 
     
     
         7 . The nucleic acid of  claim 1 , wherein the gene of interest is trfA. 
     
     
         8 . A host cell comprising the nucleic acid of  claim 1 . 
     
     
         9 . The host cell of  claim 8  further comprising a lacI gene. 
     
     
         10 . The host cell of  claim 9 , wherein the lacI gene is a mutant lacI allele. 
     
     
         11 . The host cell of  claim 10 , wherein the mutant lacI allele encodes a mutant LacI repressor protein that is capable of binding a lac operator with increased affinity. 
     
     
         12 . The host cell of  claim 8  further comprising an inactive lacZ gene. 
     
     
         13 . A method of expressing a gene of interest comprising:
 (a) providing the host cell of  claim 8 ;   (b) contacting the host cell with a LacI allosteric effector,   
       wherein the LacI allosteric effector leads to expression of the gene of interest. 
     
     
         14 . The method of  claim 13  wherein the LacI allosteric effector is derived from lactose. 
     
     
         15 . The method of  claim 13  wherein the LacI allosteric effector is a lactose analog. 
     
     
         16 . The method of  claim 15  wherein the lactose analog is isopropyl-β-D-thiogalactopyranoside. 
     
     
         17 . A method of cloning a first nucleic acid, comprising:
 (a) providing a host cell, wherein the host cell comprises recA;   (b) providing a first nucleic acid, wherein the first nucleic acid is circular;   (c) providing a second nucleic acid; and   (d) contacting the first nucleic acid with the second nucleic in the host cell, wherein first nucleic acid recombines with the second nucleic acid.   
     
     
         18 . The method of  claim 17 , wherein the recA is located in the host cell genome. 
     
     
         19 . The method of  claim 17 , wherein the recA is located on a plasmid. 
     
     
         20 . The method of  claim 19 , wherein the recA plasmid is removed after the first nucleic acid and second nucleic acid recombine. 
     
     
         21 . The method of  claim 17 , wherein the first nucleic acid comprises at least two regions of sequence identity to regions on the second nucleic acid. 
     
     
         22 . The method of  claim 17 , wherein the second nucleic acid is a host cell chromosome. 
     
     
         23 . The method of  claim 17 , wherein the first nucleic acid is a plasmid. 
     
     
         24 . The method of  claim 23 , wherein the plasmid comprises a selectable marker. 
     
     
         25 . The method of  claim 24 , wherein the selectable marker conveys kanamycin resistance. 
     
     
         26 . The method of  claim 25 , wherein the selectable marker further green fluorescent protein. 
     
     
         27 . The method of  claim 17 , wherein the host cell is a gram-negative bacterium. 
     
     
         28 . The method of  claim 27 , wherein the bacterium is  E. coli.